PROJECT SUMMARY A non-catalytic function of the mitotic kinase Aurora A (AurA) is to provide a stabilizing scaffold for N-Myc, the protein product of the MYCN gene, which is heavily amplified in many childhood neuroblastomas (NB) and late- stage neuroendocrine prostate cancers (NEPC). A strategy to block growth of cells harboring MYCN- amplification is to disrupt the complex formation of N-Myc and AurA, as this complex protects N-Myc from ubiquitin-mediated proteolysis. However, there are no approved compounds that can effectively dissociate this complex. N-Myc binds across a highly dynamic part of AurA, called the activation loop (a-loop), stabilizing the kinase in an active conformation called ?DFG-in?. In contrast, the investigational inhibitor alisertib stabilizes AurA in an inactive kinase conformation, called ?DFG-out?, in which the a-loop is moved into a position that is incompatible with N-Myc binding. It was shown in a recent paper that alisertib, and other Aurora inhibitors, have only a small energetic preference for the DFG-out state of AurA. These results likely explain why alisertib does not effectively disrupt the interaction between AurA and N-Myc in cells. This proposal outlines the use of a solution-based Frster resonance energy transfer (FRET) assay which can directly detect and quantify inhibitor preference for the DFG-in and DFG-out conformations of AurA by tracking dynamic movements of the activation loop. This proposal aims to use this FRET method to 1) define the conformational preference and cooperativity of existing ATP-competitive Aurora inhibitors with N-Myc protein, and 2) identify novel allosteric AurA inhibitors that efficiently disrupt the AurA:N-Myc complex through directed high- throughput screening (HTS). In published work, the activator of AurA, Tpx2, which shares an overlapping allosteric binding mode with N-Myc, overrides the conformational preference of most DFG-out inhibitors. Quantification of the conformational interplay between ATP-competitive inhibitors and N-Myc via a similar methodology would identify which existing Aurora inhibitors might effectively disrupt the AurA:N-Myc complex. In this proposal, the conformational preferences of Aurora inhibitors will be quantitatively compared with the DFG-in preference of N-Myc, and will then be evaluated for their ability to disrupt the AurA:N-Myc complex in a NEPC cell-line. Next, a novel HTS design will be performed to identify novel, allosteric inhibitors of AurA that induce a conformation which will block N-Myc binding, and can be used alone or in conjunction with conventional inhibitors to more-completely block N-Myc. In this HTS design, orthosteric binding is blocked at the active site with an ATP-competitive compound, Hesperadin, and ?hits? will be identified that bind to induce a conformational change to the DFG-out state through allosteric effects. Taken together, the aims outlined in this proposal will broadly-impact the kinase field, by introducing a conformationally-driven screening methodology that can be used in-solution to target specific protein complexes that drive disease. Success of these aims will identify conformationally-selective, allosteric inhibitors that represent a potentially superior method of AurA inhibition.